Various different techniques for the analysis and identification of microbiological organisms such as bacterial or fungal colonies have been developed. For example, the technique of culture collection has been established for many years. Here, a sample of material to be identified/analysed is collected and this sample is then incubated to grow a culture which can then be analysed microscopically, for example. This technique is slow (it takes at least some hours and may take days) and can miss many types of bacteria.
A second technique for microbiological analysis is so-called polymerase chain reaction (PCR). This procedure amplifies a specific region of a DNA strand. PCR diagnosis in microbiology is based upon the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of the identification of specific genes.
A further technique for microbiological analysis and identification employs a time of flight (TOF) mass spectrometer with a matrix assisted laser desorption ionization (MALDI) source. The MALDI technique was developed in the late 1980s and its application to the analysis of biological macro molecules by Tanaka at Shimadzu Corporation was awarded the Nobel Prize for Chemistry in 2002. An early description of the principles may be found in Rapid Communications in Mass Spectrometry, 1988, Volume 2, page 151, by K. Tanaka et al. Using this technique, reproducible, species-specific spectral patterns can be generated, and used to identify microorganisms at the species level.
A broad spectrum of organisms have been identified using the MALDI TOF technique, including gram-positive and gram-negative bacteria, nocardia, mycobacteria, yeasts and moulds. The technique is relatively rapid (certainly compared to culture collection techniques), has minimal consumable costs, and provides an accuracy comparable to genome sequencing. A further discussion of the MALDI TOF technique may be found in Seng, P., M. Drancourt, F. Gouriet, B. La Scola, P. E. Fournier, J. M. Rolain, and D. Raoult, “Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry” Clin. Infect. Dis. 2009, August 15; 49(4): pages 552-3; see also http://www.ncbi.nlm.nih.gov/pubmed/19583519.
Mass spectra obtained by three different research institutes, using the MALDI TOF mass spectrometry technique, for the same bacterium (in this case, E. coli (atcc 33694)), are shown in an article by Wunschel et al. in the Journal of the American Society for Mass Spectrometry, Volume 16, Issue 4, April 2005, Pages 456-462 (http://www.sciencedirect.com/science/article/pii/S1044030504008220). Each of the mass spectra shown in the Wunschel et al paper represent a 50 shot average spectrum. The Wunschel et al paper also shows generated biological fingerprints from the mass spectra of the three mass spectra obtained by the three different research institutes. These fingerprints simplify the mass spectra by, for example, removing the baseline noise. In the fingerprints of the Wunschel paper, the horizontal (x) axis represents mass to charge ratio (m/z) whilst the vertical (y) axis represents relative intensity of the peaks.
Whilst it may be seen in the Wunschel et al paper that there are clearly peaks in common between the three fingerprints (in particular, the large peak around m/z=7,000 and some smaller peaks that appear to correspond around m/z=9,500) equally there are many peaks that appear only in one or other of the three fingerprints. Since the fingerprints themselves have been generated from nominally identical microbiological materials, the accuracy of identification (by comparison of the fingerprints with a library of such fingerprints) is directly related to the degree to which the measured fingerprint corresponds with the fingerprint in the database of the microbe under analysis.
Part of the reason for the discrepancy between the three fingerprints in the Wunschel et al paper is that the MALDI TOF mass spectrometry technique currently employed generates low to very low resolution fingerprints, albeit at good sensitivity and relatively low cost. In bacterial identification, upwards of 200 peaks of bacterial origin are detected, but perhaps only a quarter of these relate to (that is, are specific to) a particular species and can thus serve to identify or differentiate that species from others.
In addition to the relatively low resolution (resolution being a measure of the ability to discriminate between adjacent peaks), current databases also contain fingerprints with m/z only up to around 10,000. However, as may be seen in the fingerprint generated from the National Institute of Standards and Technology in the Wunschel et al paper, it would be desirable to extend the mass range up to 20,000. Moreover, a higher resolution and higher sensitivity would allow for a more specific identification.
The current MALDI TOF for bacteria identification mainly uses linear TOF mass spectrometers. High resolution instruments do exist. For example, devices such as multi-reflection TOFs with ion mirrors are known as such. However, they are expensive and large and are inherently less sensitive than existing linear TOF mass spectrometers employed for biological identification. The FTMS instruments such as the Orbitrap™ and FT-ICR MS instruments can provide very high sensitivity but have limitations on their mass range and are not suited to the larger singly charged species typically produced by a MALDI ion source.